The aim of plant biotechnology is the generation of plants with advantageous novel properties, such as pest and disease resistance, resistance to environmental stress (e.g., drought), improved qualities (e.g., high yield), or for the production of certain chemicals or pharmaceuticals. Appropriate gene expression rates play an important role in order to obtain the desired phenotypes. The gene expression rate is mainly modulated by the promoter, additional DNA sequence located in the 5′ untranscribed and 5′ untranslated region and the terminator sequences of a given gene. Promoters are the portion of DNA sequences located at the 5′ end a gene which contains signals for RNA polymerases to begin transcription so that a protein synthesis can then proceed. Regulatory DNA sequences positioned in the 5′ untranscribed region modulate gene expression in response to specific biotic (e.g. pathogen infection) or abiotic (e.g. salt-, heat-, drought-stress) stimuli. Furthermore, other so called “enhancer” sequences have been identified that elevate the expression level of nearby located genes in a position and orientation independent manner.
Beside the elements located on the untranscribed regions of a gene (e.g. promoter, enhancer), it is documented in a broad range of organisms (e.g. nematodes, insects, mammals and plants) that some introns have gene expression enhancing properties. In plants, the inclusion of some introns in gene constructs leads to increased mRNA and protein accumulation relative to constructs lacking the intron. This effect has been termed “intron mediated enhancement” (IME) of gene expression (Mascarenhas et al., (1990) Plant Mol. Biol. 15:913-920). Introns known to stimulate expression in plants have been identified in maize genes (e.g. tubA1, Adh1, Sh1, Ubi1 (Jeon et al. (2000) Plant Physiol. 123:1005-1014; Callis et al. (1987) Genes Dev. 1:1183-1200; Vasil et al. (1989) Plant Physiol 91:1575-1579; Christiansen et al., (1992) Plant Mol. Biol. 18:675-6891) and in rice genes (e.g. salT, tpi [McElroy et al., (1990) Plant Cell 2: 163-171; Xu et al. (1994) Plant Physiol 106:459-467]). Similarly, introns from dicotyledonous plant genes like those from petunia (e.g. rbcS), potato (e.g. st-ls1) and from Arabidopsis thaliana (e.g. ubq3 and pat1) have been found to elevate gene expression rates (Dean et al., (1989) Plant Cell 1:201-208; Leon et al. (1991) Plant Phyisiol. 95:968-972; Norris et al. (1993) Plant Mol Biol 21:895-906; Rose and Last (1997) Plant J 11:455-464). It has been shown that deletions or mutations within the splice sites of an intron reduce gene expression, indicating that splicing might be needed for IME (Mascarenhas et al. (1990) Plant Mol Biol 15:913-920; Clancy and Hannah (2002) Plant Physiol 130:918-929). However, that splicing per se is not required for a certain IME in dicotyledonous plants has been shown by point mutations within the splice sites of the pat1 gene from A. thaliana (Rose and Beliakoff (2000) Plant Physiol 122:535-542).
Enhancement of gene expression by introns is not a general phenomenon because some intron insertions into recombinant expression cassettes fail to enhance expression (e.g. introns from dicot genes (rbcS gene from pea, phaseolin gene from bean and the stls-1 gene from Solanum tuberosum) and introns from maize genes (adh1 gene the ninth intron, hsp81 gene the first intron)) (Chee et al. (1986) Gene 41:47-57; Kuhlemeier et al. (1988) Mol Gen Genet. 212:405-411; Mascarenhas et al. (1990) Plant Mol Biol 15:913-920; Sinibaldi and Mettler (1992) In W E Cohn, K Moldave, eds, Progress in Nucleic Acid Research and Molecular Biology, Vol 42. Academic Press, New York, pp 229-257; Vancanneyt et al., 1990 Mol Gen Gent 220:245-250). Therefore, not each intron can be employed in order to manipulate the gene expression level of alien genes or endogenous genes in transgenic plants. What characteristics or specific sequence features must be present in an intron sequence in order to enhance the expression rate of a given gene is not known in the prior art and therefore from the prior art it is not possible to predict whether a given plant intron, when used heterologously, will cause IME.
The introduction of a foreign gene into a new plant host does not always result in a high expression of the incoming gene. Furthermore, if dealing with complex traits, it is sometimes necessary to modulate several genes with spatially or temporarily different expression pattern. Introns can principally provide such modulation. However multiple use of the same intron in one plant has shown to exhibit disadvantages. In those cases it is necessary to have a collection of basic control elements for the construction of appropriate recombinant DNA elements. However, the available collection of introns with expression enhancing properties is limited and alternatives are needed.
Thus, there is still a growing demand for basic control elements including promoters, regulatory sequences (e.g., inducible elements, enhancers) or intron sequences that have an impact on gene expression rates. It is therefore an objective of the present invention, to provide a highly reproducible and reliable method for the identification of introns with expression enhancing properties.
This objective is achieved by the methods provided within this invention.